ATPase copper transporting beta attenuates malignant features with high expression as an indicator of favorable prognosis in breast cancer

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ATPase copper transporting beta (ATP7B) functions as a copper-transporting ATPase that ejects copper from cells. Although high expression of ATP7B has been reported to increase cisplatin resistance, its role in breast cancer (BC) remains unclear. This study aimed to elucidate the function of ATP7B in BC cells and its significance in patients with BC. Methods. The mRNA and protein expression levels of ATP7B were evaluated in BC and non-cancerous mammary cell lines. Polymerase chain reaction (PCR) array analysis was conducted to determine the correlation between ATP7B and 84 cancer-related genes. ATP7B knockdown was performed using small interfering RNA, and cell proliferation, invasiveness, and migration were analyzed. The associations between the mRNA and protein expression of ATP7B and clinicopathological factors were also investigated in 156 patients with BC. Results. ATP7B was found to be highly expressed in estrogen receptor-positive and human epidermal growth factor receptor 2-positive BC cell lines. PCR array analysis revealed a significant correlation between the expression level of ATP7B and those of cadherin 1, estrogen receptor 1, and MET proto-oncogene. ATP7B knockdown significantly increased the proliferation, invasiveness, and migration of MDA-MB-361 and MDA-MB-415 cells. Patients with high ATP7B expression at the mRNA and protein levels experienced favorable prognoses. In addition, ATP7B expression level was identified as an independent prognostic factor in multivariate analysis. Conclusions. ATP7B is involved in promoting anti-cancer activities of tumor suppressors in BC cells across different subtypes and is considered a prognostic marker for BC. ATPase copper transporting beta breast cancer estrogen receptor prognostic marker Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Breast cancer (BC) is the most common cancer in women worldwide[ 1 ]. BC is classified via immunohistochemical analysis using conventional targets, including the estrogen receptor (ER), progesterone receptor (PgR), and human epidermal growth factor receptor 2 (HER2), and is treated according to the classification. Although several therapeutic agents are available and are still being developed, the 5-year survival rate of patients with distant metastases is as low as 27%, indicating that curing BC remains difficult [ 1 ]. Therefore, new biomarkers and therapeutic target molecules are needed to improve the prognosis of patients with BC. ATPases are a general term for enzymes that hydrolyze the phosphate bonds of adenosine triphosphate and convert the energy obtained by hydrolysis to other tasks. The function of P-type ATPases is the transport of various ions and lipids [ 2 ]. One of the P-type ATPases, copper-transporting ATPase α/β (ATP7A/ATP7B), is involved in the intracellular transport and homeostasis of copper. Mutations in ATP7B are known to cause Wilson's disease as biliary excretion of copper is inhibited due to these mutations [ 3 ]. In malignant tumors, high expression of ATP7A or ATP7B increases cisplatin resistance [ 4 , 5 ]. Recently, ATP7A and ATP7B have also attracted attention owing to their potential involvement in cuproptosis [ 6 ]. ATP7A contributes to cisplatin resistance by regulating miRNAs in BC cell lines [ 7 ]. High expression of ATP7B was found to be associated with decreased survival in patients with colorectal and lung squamous cell carcinomas and increased survival in those with renal clear cell carcinoma, low-grade glioma, and thyroid cancer [ 8 ]. Although BC cell lines expressing ATP7B have been reported to be more resistant to cisplatin than those without its expression[ 9 ], the functional role of ATP7B in BC and its impact on patients have not been reported to date . This study aimed to elucidate the role of ATP7B in BC cells and determine the significance of its expression in patients with BC. Materials and Methods Ethics This study was conducted in accordance with the principles of the Declaration of Helsinki and was approved by the Institutional Review Board and Ethics Committee of Nagoya University Hospital (approval no.: 2019-0028). All patients provided written informed consent for the use of clinical specimens and data. Sample collection A total of 13 BC cell lines (BT-20, BT-474, BT-549, HCC1419, HCC1954, Hs578T, MCF7, MDA-MB-231, MDA-MB-361, MDA-MB-415, MDA-MB-468, SK-BR-3, and ZR-75-1) and two non-cancerous breast epithelial cell lines (MCF-10A and MCF-12A) were obtained. BT-549, HCC1419, HCC1954, and Hs578T cell lines were purchased from the Japanese Collection of Research Bioresources Cell Bank (Osaka, Japan). BT-474, MCF7, and MCF-12A cells were kindly provided by Prof. David Sidransky of Johns Hopkins University (Baltimore, MD, USA). Other cell lines were purchased from American Type Culture Collection (Manassas, VA, USA). All cells were stored using a cell preservation solution (Cell Banker; Mitsubishi Chemical Medicine Corporation, Tokyo, Japan) at − 80°C, cultured in RPMI-1640 (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS), and incubated in an atmosphere of 5% carbon dioxide at 37°C [ 10 , 11 ]. BC and non-BC tissues were collected from 156 patients who were pathologically diagnosed with BC and underwent breast surgery at Nagoya University Hospital between March 2002 and May 2007. Noncancerous tissue was collected at least 3 cm from the edge of the tumor. All harvested tissues were immediately cut into approximately 1.5 mm sections and stored at − 80°C [ 12 ]. The BC stages were classified using the Union for International Cancer Control (UICC) staging system (8th edition). Perioperative adjuvant therapy was determined based on the patient's general condition, pathology, subtype classification, and shared decision-making between the attending physician and patient [ 12 ]. Quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR) ATP7B mRNA expression levels were determined using RT-qPCR. RNA was extracted from the BC and non-cancerous specimens collected from 156 patients and each cell line (8.0 x 10 6 cells per cell line). cDNA was synthesized as previously described [ 10 , 11 ]. Glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ) mRNA levels were quantified to normalize the expression levels. The specific primers for each gene were as follows: ATP7B , forward 5'-AGATCACAGCCAGAGAAGGG-3' and reverse 5'-GCCAACATTGTCAAAAGCAA-3', which generated a 110-bp product; and GAPDH , forward 5'-GAAGGTGAAGGTCGGAGTC-3' and reverse 5'-GAAGATGGTGATGGGATTTC-3', which generated a 226-bp product [ 12 ]. qRT-PCR was performed using an ABI StepOnePlus real-time PCR System (Applied Biosystems, Foster City, CA, USA), as previously described [ 10 , 11 ]. The mRNA expression level of ATP7B was determined by dividing the value of each sample by the corresponding GAPDH value [ 10 , 11 ]. PCR array analysis To determine the correlation between the expression levels of ATP7B and 84 cancer-related genes in BC cell lines, PCR array analysis was performed using the RT 2 Profiler PCR Array Human Oncogenes & Tumor Suppressor Genes (Qiagen, Hilden, Germany), according to the manufacturer's protocol. The relative expression levels of these genes in each sample were determined by dividing the relevant values by their corresponding GAPDH values. ATP7B knockdown using ATP7B -specific small interfering RNAs (siRNAs) MDA-MB-361 and MDA-MB-415 cell lines were transfected with siRNA specific for ATP7B ( designated “si ATP7B ”: 5'-CCAAUUGAUAUUGAGCGGUUATT-3'; Hokkaido System Science, Sapporo, Japan) to knockdown ATP7B . Fluorescein-labeled AccuTarget negative control siRNA (siControl, Cosmo Bio Co. Ltd., Tokyo, Japan) served as the nontargeting siRNA, designated “siControl.” BC cells were transfected with siRNAs via electroporation using the Neon System (Thermo Fisher Scientific, Waltham, MA, USA). The untransfected cells were electropulsed without siRNA. After the electric pulse, cells were cultured in antibiotic-free RPMI-1640 with 10% FBS for 72 h. The knockdown efficiency was determined using qRT-PCR and western blotting. Western blotting Western blotting was performed using a Wes Simple Western System (ProteinSimple, San Jose, CA, USA), according to the manufacturer’s instructions. Cultured cells were lysed in RIPA lysis buffer and the lysate was stored at − 30°C. Protein concentrations were measured using the BCA protein assay kit (Thermo Fisher Scientific). Protein samples were aliquoted into assay plates and automatically detected in individual capillaries. Anti-ATP7B antibody (1:250 dilution; cat. no. ab124973; Abcam, Cambridge, UK) and anti-beta-actin antibody (1:250 dilution; cat. no. ab6276; Abcam, Cambridge, UK) were used as the primary antibodies. Streptavidin Western horseradish peroxidase and anti-mouse or anti-rabbit secondary antibodies (ProteinSimple, San Jose, CA, USA) were selected based on the corresponding primary antibody [ 13 , 14 ]. Proliferation assay Cell proliferation was evaluated using the Cell Counting Kit-8 (CCK-8) (Dojindo Molecular Technologies, Inc., Kumamoto, Japan). MDA-MB-361 (1.0 × 10 4 cells per well) and MDA-MB-415 (1.0 × 10 4 cells per well) cells transfected with si ATP7B or siControl, or untransfected cells were seeded into 96-well plates with RPMI-1640 containing 2% FBS. Each sample was added to six wells and cultured for the indicated time periods. The optical density (450 nm) of each well was measured 2 h after the addition of 10 µL of CCK-8 solution from the start of seeding to day 5 post-seeding [ 12 ]. Invasiveness assay Cellular invasiveness was determined using BioCoat Matrigel Invasion Chambers (pore size 8‑µm; Corning Inc., Corning, NY, USA), according to the manufacturer's protocol. After transfection, MDA-MB-361 (3 × 10 5 cells per well) and MDA-MB-415 (3 × 10 5 cells per well) cells were suspended in serum-free RPMI-1640 and seeded into the upper chambers. RPMI-1640 medium supplemented with 20% FBS was added to the bottom row of the wells. After 72 h of incubation, cells on the membrane surfaces were fixed and stained with Diff Quik (cat. no. 16920; Sysmex, Kobe, Japan ) solutions I and II for 5 s at room temperature. Cells on the membrane were counted in 10 randomly selected fields of view using an upright microscope (Olympus Corporation) at × 100 magnification [ 12 ]. Migration assay The migration of MDA-MB-361 and MDA-MB-415 cells was determined using a wound-healing assay. After transfection, MDA-MB-361 (5.6 × 10 4 cells per well) and MDA-MB-415 (5.6 × 10 4 cells per well) cells were seeded in each well of Culture-Insert 2 Well (Ibidi, Martinsried, Germany), which were attached to 24-well plate using RPMI-1640 containing 10% FBS. After 24 h, the insert was removed and replaced with FBS-free RPMI-1640 medium and the 24-well plate was placed in an IncuCyte SX5 analysis system (Sartorius, Gottingen, Germany). The same sites were automatically photographed at 0, 12, 24, 48, and 72 h. Wound widths were measured 20 times per well at 100-µm intervals [ 12 ]. Immunohistochemistry Of the 156 patients mentioned above, specimens from 152 were available for immunohistochemical analysis. Formalin‑fixed, paraffin‑embedded sections (4‑µm thick) were constructed from blocks of resected specimens. The ATP7B rabbit polyclonal antibody (1:500 dilution) (cat. no. NB100-360; Novus biologicals, LLC., Centennial, CO, USA) was used for immunohistochemistry, and sections were incubated overnight at 4°C. The EnVision + System- HRP Labelled Polymer Anti-Rabbit (cat. no. K4003; Dako North America Inc. Carpinteria, CA, USA) was used as the secondary antibody and the sections were incubated for 30 min at room temperature. The cancerous area of each section was observed under an upright light microscope (Olympus Corporation; × 40, × 100, and × 400 magnification). The intensity of staining (IS) in the cytoplasm of cancer cells was evaluated and divided into four levels, ranging from 0 (negative) to 3 (strong). The percentage of staining (PS) was evaluated for whole cancers and divided into 11 levels, ranging from 0 to 100% in 10% increments. The IP score was assigned by multiplying the IS by the PS. Public Datasets of BC Cell Lines and Patients The mRNA expression levels of ATP7B in 59 BC cell lines were obtained from the Cancer Cell Line Encyclopedia (CCLE) database ( https://sites.broadinstitute.org/ccle/ ). The data were accessed on August 28, 2022. The Kaplan-Meier plotter website ( http://kmplot.com/analysis/index.php?p=background ) was used to analyze relapse-free survival (RFS) and overall survival (OS) of patients with BC based on ATP7B expression levels. Patients were divided into two groups based on their median expression levels[ 15 ]. The data were accessed on May 3, 2021. Statistical analyses Numerical variables between the two groups were compared using the Mann-Whitney test; comparisons between multiple groups were performed using ANOVA followed by Tukey's post hoc test. The correlation between ATP7B and cancer-related gene expression levels in PCR array analysis was assessed using Spearman's rank correlation test. The associations between mRNA or protein expression levels of ATP7B and clinicopathological factors were analyzed using the χ2 test. Disease-free survival (DFS) and OS were calculated using the Kaplan-Meier method, and survival curves were compared using the log-rank test. Multivariate analysis was performed using the Cox hazard model. All statistical analyses were performed using JMP 16 software (SAS Institute Inc., Cary, NC, USA), and statistical significance was defined as p < 0.05. Results ATP7B mRNA expression and its association with other cancer-related genes in BC cell lines The mRNA expression levels of ATP7B in 13 BC cell lines and two non-cancerous cell lines are shown in Fig. 1 a. The ER, PgR, and HER2 statuses of cell lines have been evaluated in previous studies [ 16 , 17 ]. ATP7B mRNA levels in ER-positive and HER2-positive cell lines were significantly higher than those in ER-negative and HER2-negative BC cells ( p = 0.015 and p = 0.028, respectively). To compensate for the small number of cell lines, ATP7B mRNA expression levels in additional BC cell lines were obtained from the CCLE database for verification. The ER, PgR, and HER2 statuses of each cell line were obtained from previous studies [ 18 – 20 ]. Among the 60 BC cells with available data regarding ATP7B expression levels, ER-positive, PgR-positive, and HER2-positive cells had significantly higher ATP7B mRNA levels than their negative counterparts ( p < 0.001, p = 0.002, and p = 0.040, respectively; Fig. 1 b). PCR array analysis revealed that ATP7B mRNA expression levels were positively correlated with those of several well-known oncogenes, such as cadherin 1 ( CDH1 ) and estrogen receptor 1 ( ESR1 ), and negatively correlated with MET proto-oncogene ( MET ) (Tables 1 and S1). Table 1 Correlations between mRNA expression levels of ATP7B and cancer-related genes. Gene Official Full Name Correlation Coefficient p -value CDH1 Cadherin 1, type 1, E-cadherin (epithelial) 0.863 < 0.001 ESR1 Estrogen receptor 1 0.709 0.007 RET Ret proto-oncogene 0.681 0.010 ZHX2 Zinc fingers and homeoboxes 2 0.632 0.021 MYB V-myb myeloblastosis viral oncogene homolog (avian) 0.615 0.025 MYCN V-myc myelocytomatosis viral related oncogene, neuroblastoma derived 0.615 0.025 MET Met proto-oncogene (hepatocyte growth factor receptor) −0.681 0.010 JUN Jun proto-oncogene, AP-1 transcription factor subunit −0.615 0.025 ETS1 V-ets erythroblastosis virus E26 oncogene homolog 1 (avian) −0.610 0.027 PML Promyelocytic leukemia −0.593 0.033 TGFB1 Transforming growth factor, beta 1 −0.566 0.044 Effects of ATP7B knockdown in BC cell lines Western blotting was performed using representative BC cell lines with high or low ATP7B mRNA expression to confirm the protein expression of ATP7B. Among these cell lines, MDA-MB-361 represents the ER-positive/HER2-positive subtype, and MDA-MB-415 represents the ER-positive/HER2-negative subtype. MDA-MB-231, one of the cell lines with the lowest ATP7B mRNA expression, was used as a negative control (Fig. 1 c). Cells transfected with siRNA expressed lower levels of ATP7B mRNA and protein (Fig. 1 d and e). To determine the oncological role of ATP7B in BC cells, cell proliferation, invasiveness, and migration were evaluated using knockdown cells. During the entire study period, proliferation was significantly enhanced in si ATP7B -transfected MDA-MB-361 and MDA-MB-415 cells compared to that in untransfected and siControl-transfected cells (Fig. 2 a). In the invasiveness assay, more si ATP7B -transfected than siControl-transfected or untransfected MDA-MB-361 and MDA-MB-415 cells passed through the Matrigel (Fig. 2 b). Moreover, the migratory abilities of MDA-MB-361 and MDA-MB-415 cells were enhanced following si ATP7B transfection (Fig. 2 c). Association between ATP7B mRNA expression levels and clinicopathological factors ATP7B mRNA expression levels were evaluated in both BC and non-cancerous specimens. The ratio of ATP7B mRNA expression levels between cancerous and non‑cancerous specimens was defined as the ‘C/N ratio.’ The mean C/N ratio (± SD) was 1.28 ± 1.64, with 69 (44.2%) patients having a C/N ratio greater than one. The ATP7B C/N ratios were not predominant in the T category, lymph node metastasis, or UICC stage (Fig. 3 a). Regarding conventional biomarkers, ER-positive specimens (n = 119) had a higher ATP7B C/N ratio than ER-negative specimens (n = 37; p < 0.001), and PgR-positive specimens (n = 108) had significantly higher ATP7B C/N ratios than PgR-negative specimens (n = 48; p = 0.003; Fig. 3 b). The ATP7B C/N ratio did not differ significantly between HER2-positive (n = 37) and HER2-negative specimens (n = 111; p = 0.279; Fig. 3 b). Patients with a C/N ratio greater than one were assigned to the “high ATP7B group” (n = 69), while those with a C/N ratio less than one were assigned to the “low ATP7B group” (n = 87). The associations between clinicopathological factors and ATP7B expression are shown in Table 2 . Tumor size, lymph node metastasis, or UICC pathological stage did not differ significantly between the two groups. The high ATP7B group had more ER-positive and PgR-positive patients than the low ATP7B group ( p = 0.002 and p = 0.030, respectively). Table 2 Associations between ATP7B mRNA expression and the clinicopathological characteristics of 156 patients with breast cancer Characteristics Expression of ATP7B p -value High ATP7B group (n = 69) Low ATP7B group (n = 87) Age (range) 50 (26–78) 54 (30–77) 0.024 a Histology 0.138 DCIS 5(7.3%) 1(1.1%) IDC 57(82.6%) 81(93.1%) ILC 4(5.8%) 2(2.3%) Other 3(4.3%) 3(3.5%) UICC T category 0.050 Tis/T1 37(53.6%) 33(37.9%) T2/T3/T4 32(46.4%) 54(62.1%) Lymph node status 0.147 Positive 30(43.5%) 48(55.2%) Negative 39(56.5%) 39(44.8%) UICC pathological stage 0.133 0/I 26(37.7%) 23(26.4%) II/III/IV 43(62.3%) 64(73.6%) ER status 0.002 a Positive 61(88.4%) 58(66.7%) Negative 8(11.6%) 29(33.3%) PgR status 0.030 a Positive 54(78.3%) 54(62.1%) Negative 15(21.7%) 33(37.9%) HER2 status 0.176 Positive 12(17.4%) 25(28.7%) Negative 52(75.4%) 59(67.8%) Unknown 5(2.2%) 3(3.5%) Adjuvant therapy 0.101 Endocrine therapy alone 28(40.6%) 22(25.3%) Chemotherapy alone 8(11.6%) 21(24.1%) Endocrine and chemotherapy 26(37.7%) 36(41.4%) None 7(10.1%) 8(9.2%) Data are expressed as the median (range) or number (%). a p <0.05. ATP7B, ATPase Copper Transporting Beta; DCIS, ductal carcinoma in situ ; ER, estrogen receptor; HER2, human epidermal growth factor 2; IDC, invasive ductal carcinoma; ILC, invasive lobular carcinoma; PgR, progesterone receptor; Tis, tumor in situ ; UICC, Union for International Cancer control The high ATP7B group had a significantly longer DFS than the low ATP7B group (5-year DFS, high ATP7B group: 92.7%, low ATP7B group: 76.6%; p = 0.004; Fig. 3 c). The OS rates in the high ATP7B group were also longer than that in the low ATP7B group (5-year OS: high ATP7B group, 92.8%; low ATP7B group, 88.4%; p = 0.019; Fig. 3 d). To compensate for the small number of patients in our cohort, the prognostic value of ATP7B expression was validated using the Kaplan-Meier plotter website. Similarly, when patients were separated based on the median ATP7B expression, the high ATP7B expression group exhibited significantly longer RFS (n = 4929; p < 0.001) and OS (n = 1879; p = 0.013) (Fig. S1 a and b). Multivariate analysis of DFS revealed ‘lymph node metastasis’ (HR, 3.56; 95% CI, 1.42–8.92; p = 0.007) and ‘low ATP7B expression’ (HR, 2.82; 95% CI, 1.15–6.92; p = 0.024) as independent prognostic factors (Table 3 ). Table 3 Prognostic factors for disease-free survival in 156 breast cancer patients. Variable n Univariate Multivariate Hazard ratio 95% CI p -value Hazard ratio 95% CI p -value Age, > 60 years 53 1.03 0.48–2.18 0.948 Tumor size, >2cm 86 3.69 1.51–8.99 0.004 a 2.27 0.91–5.68 0.081 Node status, positive 78 4.99 2.05–12.2 < 0.001 a 3.56 1.42–8.92 0.007 a ER status, negative 37 1.82 0.86–3.88 0.120 PgR status, negative 48 1.59 0.77–3.27 0.210 HER2 status, positive 37 1.93 0.92–4.06 0.082 Low ATP7B expression 87 3.66 1.50–8.93 0.004 a 2.82 1.15–6.92 0.024 a a p <0.05. Univariate analysis: Cox proportional hazards model. Multivariate analysis: Cox proportional hazards model. CI, confidence interval; ER, estrogen receptor; HER2, human epidermal growth factor 2; PgR, progesterone receptor Assessment of ATP7B protein expression status by immunohistochemistry At the protein level, cytoplasmic ATP7B staining was evaluated by immunohistochemistry in 152 BC specimens. Representative staining of the IS and PS of ATP7B is shown in Fig. 4 a. Patients with an IP score (IS x PS score) of 150 or higher were assigned to the ‘high ATP7B group’ (n = 73) while the remaining patients were assigned to the ‘low ATP7B group’ (n = 79). The ATP7B C/N ratio was higher in the high ATP7B group than in the low ATP7B group ( p = 0.026; Fig. 4 b), which validated the consistency between the mRNA and protein levels. Although DFS did not differ (Fig. 4 c), the OS rates were significantly higher in the high ATP7B group than in the low ATP7B group (5-year OS: high ATP7B group, 94.5%; low ATP7B group, 86.1%; p = 0.041; Fig. 4 d). Multivariate analysis of OS revealed ‘lymph node metastasis’ (HR, 4.71; 95% CI, 1.75–12.7; p = 0.002), ‘ER negative’ (HR, 7.92; 95% CI, 1.01–61.8; p = 0.048), and ‘low ATP7B expression’ (HR, 2.38; 95% CI, 1.02–5.57; p = 0.046) as independent prognostic factors (Table S2 ). Discussion In this study, ATP7B interfered with tumor progression by suppressing the proliferation, invasiveness, and migration of BC cells. Furthermore, the analysis of clinical specimens revealed that ATP7B mRNA and protein expression were independent prognostic factors, thus supporting the in vitro results. ATP7B is a member of the P -type ATPase family and is involved in intracellular copper transport and homeostasis [ 3 ]. Notably, ATP7B is one of the genes that has attracted considerable attention for its potential involvement in cuproptosis [ 6 ]. According to a recent study, ATP7B is differentially expressed in various carcinomas, suggesting a prognostic implication for patients with low-grade glioma and renal clear cell carcinoma [ 21 ]. ATP7B may also serve as a therapeutic target to improve the efficacy of docetaxel in prostate cancer [ 22 ]. In BC, ATP7B induces resistance to cisplatin [ 9 ]. High ATP7B expression in patients with ER-positive BC has also been reported to be associated with a lower risk of relapse [ 23 ]. However, the oncological role and significance of ATP7B in patients have not been elucidated to date . In this study, ATP7B was highly expressed in ER-positive cell lines, consistent with the results obtained from the CCLE database. Furthermore, our clinical specimens from ER-positive patients with BC were found to have high expression of ATP7B compared to those from ER-negative patients. Based on PCR array analysis, the expression level of ESR1 correlated positively with that of ATP7B , thus supporting these observational results. A previous study did not find a significant relationship between ATP7B and ER-positivity in patients with BC [ 9 ]; this finding may be due to the small number of samples (41 patients), which resulted in insufficient statistical power. The consistency of our results with cell lines and clinical samples implies that ATP7B is involved in the ER signaling pathway in BC. However, further pathway analyses are required to clarify the role of ATP7B in this regard . Overall, knockdown of ATP7B was found to promote malignant phenotypes in BC cells and patients with high ATP7B expression had a favorable prognosis in this study. The expression status of ATP7B was also identified as an independent prognostic factor at both the mRNA and protein levels, leading to more robust results. Although functional analysis of ATP7B in BC cells and the prognostic impact of its expression in patients with BC have not been previously assessed, a recent study revealed the association between cuproptosis-related gene expression and prognosis in ER-positive patients with BC [ 23 ]. Accordingly, patients with high expression of some cuproptosis-related genes, including ATP7B , had favorable relapse-free survival rates . Although these results are similar to those of our study, we have demonstrated that ATP7B alone can be used as a prognostic marker for BC, regardless of subtypes. Although functional analyses revealed that ATP7B plays tumor-suppressive roles in BC cells, elucidating the underlying mechanism remains a difficult task. Nevertheless, based on the results of our PCR array analysis of BC cells and those of previous studies, several explanations can be proposed. The highest correlation coefficient was observed between the expression levels of ATP7B and CDH1 , which encodes E-cadherin. E-cadherins contribute to the establishment and maintenance of polarized and differentiated epithelia through intercellular adhesion complexes, and their inactivation increases the metastatic capacity, which leads to poor prognoses in BC [ 24 ]. In contrast, ATP7B expression negatively correlated with MET expression. MET encodes the tyrosine kinase receptor for hepatocyte growth factor, and its activation promotes cellular invasiveness, angiogenesis, and metastasis [ 25 ]. Although no studies to date have suggested a direct relationship between ATP7B and these molecules, ATP7B may affect the expression of these genes to suppress tumor progression in BC. Notably, analysis of the clinical samples identified both the mRNA and protein levels of ATP7B as prognostic markers. The cut-off values of ATP7B expression levels have not been established to date. In this study, in terms of the mRNA expression levels, patients with a C/N ratio greater than one were assigned to the “high ATP7B group” (n = 69) while those with a C/N ratio less than one were assigned to the “low ATP7B group” (n = 87). This grouping method is considered relatively reasonable. On the other hand, for the immunohistochemistry, we grouped the patients according to the IP score. Patients with an IP score of 150 or higher were then assigned to the ‘high ATP7B group’ (n = 73) while the remaining patients were assigned to the ‘low ATP7B group’ (n = 79). This cut-off value allocated almost the same number of cases as the grouping at the mRNA expression levels. Moreover, because ATP7B mRNA expression was shown to be higher in ‘high ATP7B group’, this criterion closely reflected the grouping at the mRNA level. Although our results are supported by the public database, further validation studies will be required to determine appropriate cut-off values. This study had several limitations. As described above, the mechanism by which ATP7B is involved in tumor suppression has not been fully investigated, despite the proposal of several theories. In particular, the mechanism by which ATP7B affects copper transport in BC cells to attenuate their malignant features remains to be elucidated. Second, adjuvant medication therapy could have affected the patient prognoses in our cohort data. To compensate for this effect, we used a public database to validate our results. In addition, further studies, such as in vivo experiments, are warranted to elucidate the potential therapeutic targets of ATP7B. In conclusion, this study revealed the tumor-suppressive roles of ATP7B in BC cells. Moreover, this study highlighted that high ATP7B expression in cancerous tissues could be a favorable prognostic biomarker in patients with BC. Declarations Acknowledgements Not applicable. Funding No funding was received. Availability of Data and Materials The datasets used and/or analyzed in the current study are available from the corresponding author upon reasonable request. Author information Authors and Affiliations Department of Breast and Endocrine Surgery, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 4668560, Japan Ikumi Soeda, Masahiro Shibata, Takahiro Ichikawa, Kayoko Sugino, & Norikazu Masuda Department of Surgery, Nagoya Ekisaikai Hospital, 4-66, Shonen-cho, Nakagawa-ku, Nagoya 4548502, Japan Masahiro Shibata Department of Surgery, Komaki City Hospital, 1-20, Joubushi, Komaki Aichi 4858520, Japan Takahiro Inaishi Department of Surgery and Science, Faculty of Medicine, Academic Assembly, University of Toyama 2630 Sugitani, Toyama-shi, Toyama, 930-0194, Japan Emi Kanaya Department of Gastroenterological Surgery, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 4668560, Japan Mitsuro Kanda & Masamichi Hayashi Author Contributions IS, MS, and MK conceived and designed the study. IS, MS, TIn, and TIc conducted the experiments. IS and MS analyzed the data and wrote the manuscript. MS, TIn, and TIc contributed to the acquisition of patient data. TIn, TIc, KS, EK, MK, MH, and NM contributed to the interpretation of comprehensive data and revised the manuscript for important intellectual content. All authors have read and approved the final version of the manuscript. Ethics approval and Consent to Participate The present study was approved by the Institutional Review Board and Ethics Committee of Nagoya University Hospital (approval no. 2019‑0028). Informed consent was obtained from all patients included in the study. Patient consent for Publication The participants provided written informed consent for publication as required by the Institutional Review Board and Ethics Committee of Nagoya University Hospital. Conflict of interests Norikazu Masuda received a research grant from Chugai Pharma, Eli Lilly Japan, AstraZeneca, Pfizer, Daiichi Sankyo, MSD, Eisai, Novartis, Gilead Sciences, and Ono Pharma; received lecture fees from Chugai Pharma, Pfizer, AstraZeneca, Eli Lilly Japan, Daiichi Sankyo, and Eisai; and is a board representative of the Japan Breast Cancer Research Group (JBCRG) and a board member of the Japanese Breast Cancer Society (JBCS), the Japan Society of Clinical Oncology (JSCO), and the Japan Association of Breast Cancer Screening (JABCS), all without remuneration. The other authors declare no competing interests. References Siegel RL, Miller KD, Wagle NS, Jemal A. Cancer statistics, 2023. CA Cancer J Clin. 2023;73:17–48. Palmgren MG, Nissen P. P-type ATPases. Annu Rev Biophys. 2011;40:243–66. Polishchuk EV, Merolla A, Lichtmannegger J, Romano A, Indrieri A, Ilyechova EY et al. Activation of Autophagy, Observed in Liver Tissues From Patients With Wilson Disease and From ATP7B-Deficient Animals, Protects Hepatocytes From Copper-Induced Apoptosis. Gastroenterology. 2019; 156: 1173-89 e5. Li YQ, Yin JY, Liu ZQ, Li XP. Copper efflux transporters ATP7A and ATP7B: Novel biomarkers for platinum drug resistance and targets for therapy. IUBMB Life. 2018;70:183–91. Inesi G, Pilankatta R, Tadini-Buoninsegni F. Biochemical characterization of P-type copper ATPases. Biochem J. 2014;463:167–76. Tsvetkov P, Coy S, Petrova B, Dreishpoon M, Verma A, Abdusamad M, et al. Copper induces cell death by targeting lipoylated TCA cycle proteins. Science. 2022;375:1254–61. Yu Z, Cao W, Ren Y, Zhang Q, Liu J. ATPase copper transporter A, negatively regulated by miR-148a-3p, contributes to cisplatin resistance in breast cancer cells. Clin Transl Med. 2020;10:57–73. Li Y, Yu Z. Pan-cancer analysis reveals copper transporters as promising potential targets. Heliyon. 2024;10:e37007. Kanzaki A, Toi M, Neamati N, Miyashita H, Oubu M, Nakayama K, et al. Copper-transporting P-type adenosine triphosphatase (ATP7B) is expressed in human breast carcinoma. Jpn J Cancer Res. 2002;93:70–7. Inaishi T, Shibata M, Ichikawa T, Kanda M, Hayashi M, Soeda I et al. Platelet isoform of phosphofructokinase accelerates malignant features in breast cancer. Oncol Rep. 2022; 47. Watanabe M, Shibata M, Inaishi T, Ichikawa T, Soeda I, Miyajima N, et al. MZB1 expression indicates poor prognosis in estrogen receptor-positive breast cancer. Oncol Lett. 2020;20:198. Shibata M, Kanda M, Tanaka H, Umeda S, Miwa T, Shimizu D, et al. Overexpression of Derlin 3 is associated with malignant phenotype of breast cancer cells. Oncol Rep. 2017;38:1760–6. Harris VM. Protein detection by Simple Western™ analysis. Methods Mol Biol. 2015;1312:465–8. Fujita M, Somasundaram V, Basudhar D, Cheng RYS, Ridnour LA, Higuchi H, et al. Role of nitric oxide in pancreatic cancer cells exhibiting the invasive phenotype. Redox Biol. 2019;22:101158. Györffy B, Lanczky A, Eklund AC, Denkert C, Budczies J, Li Q, et al. An online survival analysis tool to rapidly assess the effect of 22,277 genes on breast cancer prognosis using microarray data of 1,809 patients. Breast Cancer Res Treat. 2010;123:725–31. Subik K, Lee JF, Baxter L, Strzepek T, Costello D, Crowley P, et al. The Expression Patterns of ER, PR, HER2, CK5/6, EGFR, Ki-67 and AR by Immunohistochemical Analysis in Breast Cancer Cell Lines. Breast Cancer (Auckl). 2010;4:35–41. Riaz M, van Jaarsveld MT, Hollestelle A, Prager-van der Smissen WJ, Heine AA, Boersma AW, et al. miRNA expression profiling of 51 human breast cancer cell lines reveals subtype and driver mutation-specific miRNAs. Breast Cancer Res. 2013;15:R33. Dai X, Cheng H, Bai Z, Li J. Breast Cancer Cell Line Classification and Its Relevance with Breast Tumor Subtyping. J Cancer. 2017;8:3131–41. Hongisto V, Jernström S, Fey V, Mpindi JP, Kleivi Sahlberg K, Kallioniemi O, et al. High-throughput 3D screening reveals differences in drug sensitivities between culture models of JIMT1 breast cancer cells. PLoS ONE. 2013;8:e77232. Neve RM, Chin K, Fridlyand J, Yeh J, Baehner FL, Fevr T, et al. A collection of breast cancer cell lines for the study of functionally distinct cancer subtypes. Cancer Cell. 2006;10:515–27. Zhang Z, Zhang A, Shi Y, Zhao Z, Zhao Z. An association between ATP7B expression and human cancer prognosis and immunotherapy: a pan-cancer perspective. BMC Med Genomics. 2023;16:307. Song L, Nguyen V, Xie J, Jia S, Chang CJ, Uchio E, et al. ATPase Copper Transporting Beta (ATP7B) Is a Novel Target for Improving the Therapeutic Efficacy of Docetaxel by Disulfiram/Copper in Human Prostate Cancer. Mol Cancer Ther. 2024;23:854–63. Fan Y, Luo C, Wang Y, Wang Z, Wang C, Zhong X, et al. A nomogram based on cuproptosis-related genes predicts 7-year relapse-free survival in patients with estrogen receptor-positive early breast cancer. Front Oncol. 2023;13:1111480. Corso G, Figueiredo J, De Angelis SP, Corso F, Girardi A, Pereira J, et al. E-cadherin deregulation in breast cancer. J Cell Mol Med. 2020;24:5930–6. Gastaldi S, Comoglio PM, Trusolino L. The Met oncogene and basal-like breast cancer: another culprit to watch out for? Breast Cancer Res. 2010;12:208. Supplementary Files SupplementaryFigure1.pdf SupplementaryTable1.pdf SupplementaryTable2.pdf Cite Share Download PDF Status: Published Journal Publication published 02 May, 2025 Read the published version in Breast Cancer → Version 1 posted Editorial decision: Accept 14 Apr, 2025 Reviewers agreed at journal 10 Apr, 2025 Reviewers invited by journal 09 Apr, 2025 Editor assigned by journal 09 Apr, 2025 First submitted to journal 09 Apr, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6101234","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":440908990,"identity":"6462e3cb-95e2-4b69-8ae1-a9830ee34c1e","order_by":0,"name":"Ikumi Soeda","email":"","orcid":"","institution":"Nagoya Daigaku","correspondingAuthor":false,"prefix":"","firstName":"Ikumi","middleName":"","lastName":"Soeda","suffix":""},{"id":440908991,"identity":"168ddfc5-8366-42e3-9a64-f9819546bee4","order_by":1,"name":"Masahiro Shibata","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0003-1028-2796","institution":"Nagoya Daigaku","correspondingAuthor":true,"prefix":"","firstName":"Masahiro","middleName":"","lastName":"Shibata","suffix":""},{"id":440908992,"identity":"0b64e3ce-f804-4d59-8389-eee62caf0c9d","order_by":2,"name":"Takahiro Inaishi","email":"","orcid":"","institution":"Nagoya Daigaku","correspondingAuthor":false,"prefix":"","firstName":"Takahiro","middleName":"","lastName":"Inaishi","suffix":""},{"id":440908993,"identity":"02e78994-1ead-42e1-bd4c-2f71cdcc7414","order_by":3,"name":"Takahiro Ichikawa","email":"","orcid":"","institution":"Nagoya Daigaku","correspondingAuthor":false,"prefix":"","firstName":"Takahiro","middleName":"","lastName":"Ichikawa","suffix":""},{"id":440908994,"identity":"c1a5b827-7b94-4f81-a130-989eadef3617","order_by":4,"name":"Kayoko Sugino","email":"","orcid":"","institution":"Nagoya Daigaku","correspondingAuthor":false,"prefix":"","firstName":"Kayoko","middleName":"","lastName":"Sugino","suffix":""},{"id":440908995,"identity":"ec5dbf38-866c-4a74-b147-2242d637592a","order_by":5,"name":"Emi Kanaya","email":"","orcid":"","institution":"Toyama Daigaku","correspondingAuthor":false,"prefix":"","firstName":"Emi","middleName":"","lastName":"Kanaya","suffix":""},{"id":440908996,"identity":"54df8094-52b9-495a-abc5-47d8deec2155","order_by":6,"name":"Mitsuro Kanda","email":"","orcid":"","institution":"Nagoya Daigaku","correspondingAuthor":false,"prefix":"","firstName":"Mitsuro","middleName":"","lastName":"Kanda","suffix":""},{"id":440908997,"identity":"10562b46-9ec3-4df2-9b05-a8249b9d0f4a","order_by":7,"name":"Masamichi Hayashi","email":"","orcid":"","institution":"Nagoya Daigaku","correspondingAuthor":false,"prefix":"","firstName":"Masamichi","middleName":"","lastName":"Hayashi","suffix":""},{"id":440908998,"identity":"532e6f83-22bb-46d1-9ddf-2bea9ee97572","order_by":8,"name":"Norikazu Masuda","email":"","orcid":"","institution":"Nagoya Daigaku","correspondingAuthor":false,"prefix":"","firstName":"Norikazu","middleName":"","lastName":"Masuda","suffix":""}],"badges":[],"createdAt":"2025-02-25 04:16:02","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6101234/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6101234/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s12282-025-01705-7","type":"published","date":"2025-05-02T15:57:37+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":80801663,"identity":"fca32dde-7855-4278-b0f2-c272e33dc289","added_by":"auto","created_at":"2025-04-17 08:39:55","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":54742,"visible":true,"origin":"","legend":"\u003cp\u003eATP7B expression in BC cell line. \u003cstrong\u003ea.\u003c/strong\u003e \u003cem\u003eATP7B \u003c/em\u003emRNA expression in 13 BC and two non-cancerous cell lines. Error bars, mean ± SEM. \u003cstrong\u003eb.\u003c/strong\u003e Association between \u003cem\u003eATP7B\u003c/em\u003e mRNA expression levels and the status of conventional biomarkers from CCLE data. ER-positive cells, PgR-positive cells, and HER2-positive cells had significantly higher \u003cem\u003eATP7B\u003c/em\u003e mRNA levels than their negative counterparts. \u003cstrong\u003ec.\u003c/strong\u003e ATP7B expression in representative BC cell lines. ATP7B expression was \u003cu\u003edetected\u003c/u\u003e in MDA-MB-361 and MDA-MB-415, whereas ATP7B was not detected in MDA-MB-231 cells. \u003cstrong\u003ed.\u003c/strong\u003e qRT-PCR analysis of \u003cem\u003eATP7B\u003c/em\u003e mRNA expression levels after knockdown in MDA-MB-361 and MDA-MB-415 cell lines. \u003cstrong\u003ee.\u003c/strong\u003e Western blot analysis revealed inhibition of ATP7B after knockdown in MDA-MB-361 and MDA-MB-415 cell lines. ATP7B, ATPase copper transforming beta; BC, breast cancer; ER, estrogen receptor; HER2, human epidermal growth factor receptor 2; si, small interfering. \u003csup\u003e＊\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e＊＊\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01,\u003csup\u003e ＊＊＊\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-6101234/v1/d0cfc795cb6df071ff08fbdc.png"},{"id":80801648,"identity":"e2776993-e59c-4ecc-8e40-0a839b1fcb19","added_by":"auto","created_at":"2025-04-17 08:39:54","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":179106,"visible":true,"origin":"","legend":"\u003cp\u003eFunctional analysis in BC cell lines using knockdown cells. \u003cstrong\u003ea.\u003c/strong\u003e Proliferation assay: si\u003cem\u003eATP7B\u003c/em\u003e-transfected cell \u003cu\u003eproliferation was\u003c/u\u003e significantly enhanced compared to untransfected and siControl-transfected cells. \u003cstrong\u003eb.\u003c/strong\u003e Invasiveness assay: ATP7B \u003cu\u003eknockdown\u003c/u\u003e in BC cells significantly increased the number of invading cells. \u003cstrong\u003ec \u003c/strong\u003eMigration assay: the migration ability of MDA-MB-361 and MDA-MB-415 cells was enhanced after si\u003cem\u003eATP7B\u003c/em\u003e transfection. Error bars mean ± SEM. ATP7B, ATPase copper transporting beta; BC, breast cancer; si, small interfering. \u003csup\u003e＊\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e＊＊\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, \u003csup\u003e＊＊＊\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-6101234/v1/d51dc700657e2ab8223fb30f.png"},{"id":80801646,"identity":"69c0281f-1572-421e-8af4-2c54f0db28da","added_by":"auto","created_at":"2025-04-17 08:39:54","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":43420,"visible":true,"origin":"","legend":"\u003cp\u003eAssociation between ATP7B mRNA expression levels and clinicopathological factors. \u003cstrong\u003ea.\u003c/strong\u003e \u003cem\u003eATP7B\u003c/em\u003e mRNA expression levels \u003cu\u003edid not differ\u003c/u\u003e according to T-category, lymph node metastasis, or UICC stage. \u003cstrong\u003eb.\u003c/strong\u003e ER-positive and PgR-positive \u003cu\u003esamples\u003c/u\u003e had significantly higher \u003cem\u003eATP7B\u003c/em\u003e mRNA expression levels than ER-negative and PgR-negative \u003cu\u003esamples\u003c/u\u003e; however, no significant difference was found between HER2-positive and HER2-negative specimens. \u003cstrong\u003ec.\u003c/strong\u003e High \u003cem\u003eATP7B\u003c/em\u003e group experienced longer DFS than the low \u003cem\u003eATP7B\u003c/em\u003e group. \u003cstrong\u003ed.\u003c/strong\u003e \u003cu\u003eThe\u003c/u\u003e OS rates in the high \u003cem\u003eATP7B\u003c/em\u003e group were significantly longer than those in the low \u003cem\u003eATP7B \u003c/em\u003egroup. \u003csup\u003ea \u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05; ATP7B, ATPase copper transporting beta; DFS, disease‑free survival; ER, estrogen receptor; HER2, human epidermal growth factor receptor 2; OS, overall survival; PgR, progesterone receptor; Tis, carcinoma in situ; UICC, Union for International Cancer Control\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-6101234/v1/d822a98ca5c94ad06f101e76.png"},{"id":80801647,"identity":"149b8e0f-30ce-4ef7-9a73-4fd4a4193249","added_by":"auto","created_at":"2025-04-17 08:39:54","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":376012,"visible":true,"origin":"","legend":"\u003cp\u003eAssessment of ATP7B protein expression status \u003cu\u003eby\u003c/u\u003e immunohistochemistry. \u003cstrong\u003ea.\u003c/strong\u003e Representative staining for the IS and PS of ATP7B. \u003cstrong\u003eb.\u003c/strong\u003e \u003cem\u003eATP7B\u003c/em\u003e C/N ratio was higher in the high ATP7B group than in the low ATP7B group \u003cstrong\u003ec.\u003c/strong\u003e No difference in DFS was found. \u003cstrong\u003ed.\u003c/strong\u003e \u003cu\u003eThe\u003c/u\u003e OS rates in the high ATP7B group were significantly longer than those in the low ATP7B group. \u003csup\u003ea \u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05; ATP7B, ATPase copper transporting beta; DFS, disease‑free survival; IS, intensity of staining; OS, overall survival; PS, percentage of staining\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-6101234/v1/d4cfd79ac207a8a397d46870.png"},{"id":81987774,"identity":"105c45bf-6470-4109-99d0-0d51b0b18d34","added_by":"auto","created_at":"2025-05-05 16:05:56","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1819048,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6101234/v1/4783f456-cd0a-4fb4-aa3e-2b7f9ca1370a.pdf"},{"id":80801726,"identity":"80b08559-bbd8-4479-9559-f0310494b9f8","added_by":"auto","created_at":"2025-04-17 08:40:00","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":83287,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6101234/v1/d98521706f3a0c4864e15e93.pdf"},{"id":80802115,"identity":"26106095-9905-4355-8ddd-8524b367281d","added_by":"auto","created_at":"2025-04-17 08:47:54","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":228127,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6101234/v1/f684ec76d7d32b756b8d2bf8.pdf"},{"id":80801655,"identity":"068a2651-d041-43a6-91b3-3b6ccd752dfe","added_by":"auto","created_at":"2025-04-17 08:39:55","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":123247,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6101234/v1/ad2ebe198cb404a17755c0ad.pdf"}],"financialInterests":"","formattedTitle":"ATPase copper transporting beta attenuates malignant features with high expression as an indicator of favorable prognosis in breast cancer","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBreast cancer (BC) is the most common cancer in women worldwide[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. BC is classified via immunohistochemical analysis using conventional targets, including the estrogen receptor (ER), progesterone receptor (PgR), and human epidermal growth factor receptor 2 (HER2), and is treated according to the classification. Although several therapeutic agents are available and are still being developed, the 5-year survival rate of patients with distant metastases is as low as 27%, \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eindicating that curing BC remains difficult\u003c/span\u003e [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Therefore, new biomarkers and therapeutic target molecules are needed to improve the prognosis of patients with BC.\u003c/p\u003e \u003cp\u003eATPases are a general term for enzymes that hydrolyze the phosphate bonds of adenosine triphosphate and convert the energy obtained by hydrolysis to other tasks. \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eThe function of P-type ATPases is the transport of\u003c/span\u003e various ions and lipids [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. One of the P-type ATPases, copper-transporting ATPase α/β (ATP7A/ATP7B), is involved in the intracellular transport and homeostasis of copper. Mutations in ATP7B are known to cause Wilson's disease as biliary excretion of copper is inhibited due to these mutations [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. In malignant tumors, high expression of \u003cem\u003eATP7A\u003c/em\u003e or \u003cem\u003eATP7B\u003c/em\u003e increases cisplatin resistance [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Recently, \u003cem\u003eATP7A\u003c/em\u003e and \u003cem\u003eATP7B\u003c/em\u003e have also attracted attention owing to their potential involvement in cuproptosis [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. ATP7A contributes to cisplatin resistance by regulating miRNAs in BC cell lines [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. High expression of \u003cem\u003eATP7B\u003c/em\u003e was found to be associated with decreased survival in patients with colorectal and lung squamous cell carcinomas and increased survival in those with renal clear cell carcinoma, low-grade glioma, and thyroid cancer [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Although BC cell lines expressing ATP7B have been reported to be more resistant to cisplatin than those without its expression[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], the functional role of ATP7B in BC and its impact on patients have not been reported \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eto date\u003c/span\u003e. This study \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eaimed\u003c/span\u003e to elucidate the role of ATP7B in BC cells and determine the significance of its expression in patients with BC.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eEthics\u003c/h2\u003e \u003cp\u003e This study was conducted in accordance with the principles of the Declaration of Helsinki and was approved by the Institutional Review Board and Ethics Committee of Nagoya University Hospital (approval no.: 2019-0028). All patients provided written informed consent for the use of clinical specimens and data.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSample collection\u003c/h3\u003e\n\u003cp\u003eA total of 13 BC cell lines (BT-20, BT-474, BT-549, HCC1419, HCC1954, Hs578T, MCF7, MDA-MB-231, MDA-MB-361, MDA-MB-415, MDA-MB-468, SK-BR-3, and ZR-75-1) and two non-cancerous breast epithelial cell lines (MCF-10A and MCF-12A) were obtained. BT-549, HCC1419, HCC1954, and Hs578T cell lines were purchased from the Japanese Collection of Research Bioresources Cell Bank (Osaka, Japan). BT-474, MCF7, and MCF-12A cells were kindly provided by Prof. David Sidransky of Johns Hopkins University (Baltimore, MD, USA). Other cell lines were purchased from American Type Culture Collection (Manassas, VA, USA). All cells were stored using a cell preservation solution (Cell Banker; Mitsubishi Chemical Medicine Corporation, Tokyo, Japan) at \u0026minus;\u0026thinsp;80\u0026deg;C, cultured in RPMI-1640 (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS), and incubated in an atmosphere of 5% carbon dioxide at 37\u0026deg;C [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBC and non-BC tissues were collected from 156 patients who were pathologically diagnosed with BC and underwent breast surgery at Nagoya University Hospital between March 2002 and May 2007. Noncancerous tissue was collected at least 3 cm from the edge of the tumor. All harvested tissues were immediately cut into approximately 1.5 mm sections and stored at \u0026minus;\u0026thinsp;80\u0026deg;C [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eThe\u003c/span\u003e BC stages were classified using the Union for International Cancer Control (UICC) staging system (8th edition). Perioperative adjuvant therapy was determined based on the patient's general condition, pathology, subtype classification, and shared decision-making between the attending physician and patient [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eQuantitative real-time reverse transcription polymerase chain reaction (RT-qPCR)\u003c/h3\u003e\n\u003cp\u003e \u003cem\u003eATP7B\u003c/em\u003e mRNA expression levels were determined using RT-qPCR. RNA was extracted from \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ethe\u003c/span\u003e BC and non-cancerous specimens collected from 156 patients and each cell line (8.0 x 10\u003csup\u003e6\u003c/sup\u003e cells per cell line). cDNA was synthesized as previously described [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. \u003cem\u003eGlyceraldehyde-3-phosphate dehydrogenase\u003c/em\u003e (\u003cem\u003eGAPDH\u003c/em\u003e) mRNA levels were quantified to normalize the expression levels. The specific primers for each gene were as follows: \u003cem\u003eATP7B\u003c/em\u003e, forward 5'-AGATCACAGCCAGAGAAGGG-3' and reverse 5'-GCCAACATTGTCAAAAGCAA-3', which generated a 110-bp product; and \u003cem\u003eGAPDH\u003c/em\u003e, forward 5'-GAAGGTGAAGGTCGGAGTC-3' and reverse 5'-GAAGATGGTGATGGGATTTC-3', which generated a 226-bp product [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. qRT-PCR was performed using an ABI StepOnePlus real-time PCR System (Applied Biosystems, Foster City, CA, USA), as previously described [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eThe mRNA expression level of\u003c/span\u003e \u003cspan type=\"ItalicUnderline\" class=\"ItalicUnderline\" name=\"Emphasis\"\u003eATP7B\u003c/span\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ewas determined by dividing the value of each sample by the corresponding\u003c/span\u003e \u003cspan type=\"ItalicUnderline\" class=\"ItalicUnderline\" name=\"Emphasis\"\u003eGAPDH\u003c/span\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003evalue\u003c/span\u003e [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003ePCR array analysis\u003c/h3\u003e\n\u003cp\u003eTo determine the correlation between the expression levels of \u003cem\u003eATP7B\u003c/em\u003e and 84 cancer-related genes in BC cell lines, PCR array analysis was performed using the RT\u003csup\u003e2\u003c/sup\u003e Profiler PCR Array Human Oncogenes \u0026amp; Tumor Suppressor Genes (Qiagen, Hilden, Germany), according to the manufacturer's protocol. The relative expression levels of these genes in each sample were determined by dividing the relevant values by their corresponding \u003cem\u003eGAPDH\u003c/em\u003e values.\u003c/p\u003e \u003cp\u003e \u003cb\u003eATP7B knockdown using\u003c/b\u003e \u003cb\u003eATP7B\u003c/b\u003e\u003cb\u003e-specific small interfering RNAs (siRNAs)\u003c/b\u003e\u003c/p\u003e \u003cp\u003eMDA-MB-361 and MDA-MB-415 cell lines were transfected with siRNA specific for \u003cem\u003eATP7B\u003c/em\u003e (\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003edesignated\u003c/span\u003e \u0026ldquo;si\u003cem\u003eATP7B\u003c/em\u003e\u0026rdquo;: 5'-CCAAUUGAUAUUGAGCGGUUATT-3'; Hokkaido System Science, Sapporo, Japan) to knockdown \u003cem\u003eATP7B\u003c/em\u003e. Fluorescein-labeled AccuTarget negative control siRNA (siControl, Cosmo Bio Co. Ltd., Tokyo, Japan) served as the nontargeting siRNA, \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003edesignated\u003c/span\u003e \u0026ldquo;siControl.\u0026rdquo; BC cells were transfected with siRNAs via electroporation using the Neon System (Thermo Fisher Scientific, Waltham, MA, USA). The untransfected cells were electropulsed without siRNA. After the electric pulse, cells were cultured in antibiotic-free RPMI-1640 with 10% FBS for 72 h. The knockdown efficiency was determined using qRT-PCR and western blotting.\u003c/p\u003e\n\u003ch3\u003eWestern blotting\u003c/h3\u003e\n\u003cp\u003eWestern blotting was performed using a Wes Simple Western System (ProteinSimple, San Jose, CA, USA), according to the manufacturer\u0026rsquo;s instructions. Cultured cells were lysed in RIPA lysis buffer and the lysate was stored at \u0026minus;\u0026thinsp;30\u0026deg;C. Protein concentrations were measured using the BCA protein assay kit (Thermo Fisher Scientific). Protein samples were aliquoted into assay plates and automatically detected in individual capillaries. Anti-ATP7B antibody (1:250 dilution; cat. no. ab124973; Abcam, Cambridge, UK) and anti-beta-actin antibody (1:250 dilution; cat. no. ab6276; Abcam, Cambridge, UK) were used as the primary antibodies. Streptavidin Western horseradish peroxidase and anti-mouse or anti-rabbit secondary antibodies (ProteinSimple, San Jose, CA, USA) were selected \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ebased on\u003c/span\u003e the corresponding primary \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eantibody\u003c/span\u003e [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eProliferation assay\u003c/h2\u003e \u003cp\u003eCell proliferation was evaluated using the Cell Counting Kit-8 (CCK-8) (Dojindo Molecular Technologies, Inc., Kumamoto, Japan). MDA-MB-361 (1.0 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells per well) and MDA-MB-415 (1.0 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells per well) cells transfected with si\u003cem\u003eATP7B\u003c/em\u003e or siControl, or untransfected cells were seeded into 96-well plates with RPMI-1640 containing 2% FBS. Each sample was added to six wells and cultured for the indicated time periods. The optical density (450 nm) of each well was measured 2 h after the addition of 10 \u0026micro;L of CCK-8 solution from the start of seeding to day 5 post-seeding [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eInvasiveness assay\u003c/h3\u003e\n\u003cp\u003eCellular invasiveness was determined using BioCoat Matrigel Invasion Chambers (pore size 8‑\u0026micro;m; Corning Inc., Corning, NY, USA), according to the manufacturer's protocol. After transfection, MDA-MB-361 (3 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells per well) and MDA-MB-415 (3 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells per well) cells were suspended in serum-free RPMI-1640 and seeded into the upper chambers. RPMI-1640 medium supplemented with 20% FBS was added to the bottom row of the wells. After 72 h of incubation, cells on the membrane surfaces were fixed and stained with Diff Quik (cat. no. 16920; Sysmex, \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eKobe, Japan\u003c/span\u003e) solutions I and II for 5 s at room temperature. Cells on the membrane were counted in 10 randomly selected fields of view using an upright microscope (Olympus Corporation) at \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026times;\u003c/span\u003e100 magnification [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eMigration assay\u003c/h3\u003e\n\u003cp\u003eThe migration of MDA-MB-361 and MDA-MB-415 cells was determined using a wound-healing assay. After transfection, MDA-MB-361 (5.6 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells per well) and MDA-MB-415 (5.6 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells per well) cells were seeded in each well of Culture-Insert 2 Well (Ibidi, Martinsried, Germany), which were attached to 24-well plate using RPMI-1640 containing 10% FBS. After 24 h, the insert was removed and replaced with FBS-free RPMI-1640 medium and the 24-well plate was placed in an IncuCyte SX5 \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eanalysis system\u003c/span\u003e (Sartorius, Gottingen, Germany). The same sites were automatically photographed at 0, 12, 24, 48, and 72 h. Wound widths were measured 20 times per well at 100-\u0026micro;m intervals [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemistry\u003c/h2\u003e \u003cp\u003eOf the 156 patients mentioned above, specimens from 152 were available for immunohistochemical analysis. Formalin‑fixed, paraffin‑embedded sections (4‑\u0026micro;m thick) were constructed from blocks of resected specimens. The ATP7B rabbit polyclonal antibody (1:500 dilution) (cat. no. NB100-360; Novus biologicals, LLC., Centennial, CO, USA) was used for immunohistochemistry, and sections were incubated overnight at 4\u0026deg;C. The EnVision\u0026thinsp;+\u0026thinsp;System- HRP Labelled Polymer Anti-Rabbit (cat. no. K4003; Dako North America Inc. Carpinteria, CA, USA) was used as the secondary antibody and the sections were incubated for 30 min at room temperature. The cancerous area of each section was observed under an upright light microscope (Olympus Corporation; \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026times;\u003c/span\u003e40, \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026times;\u003c/span\u003e100, and \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026times;\u003c/span\u003e400 magnification). The intensity of staining (IS) in the cytoplasm of cancer cells was evaluated and divided into four levels, ranging from 0 (negative) to 3 (strong). The percentage of staining (PS) was evaluated for whole cancers and divided into 11 levels, ranging from 0 to 100% in 10% increments. The IP score was assigned by multiplying the IS by the PS.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003ePublic Datasets of BC Cell Lines and Patients\u003c/h2\u003e \u003cp\u003eThe mRNA expression levels of \u003cem\u003eATP7B\u003c/em\u003e in 59 BC cell lines were obtained from the Cancer Cell Line Encyclopedia (CCLE) database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://sites.broadinstitute.org/ccle/\u003c/span\u003e\u003cspan address=\"https://sites.broadinstitute.org/ccle/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eThe data\u003c/span\u003e were accessed on August 28, 2022. The Kaplan-Meier plotter website (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://kmplot.com/analysis/index.php?p=background\u003c/span\u003e\u003cspan address=\"http://kmplot.com/analysis/index.php?p=background\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used to analyze relapse-free survival (RFS) and overall survival (OS) of patients with BC based on \u003cem\u003eATP7B\u003c/em\u003e expression levels. Patients were divided into two groups based on their median expression levels[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The data were accessed on May 3, 2021.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analyses\u003c/h2\u003e \u003cp\u003eNumerical variables between the two groups were compared using the Mann-Whitney test; comparisons between multiple groups were performed using ANOVA followed by Tukey's post hoc test. The correlation between ATP7B and cancer-related gene expression levels in PCR array analysis was assessed using Spearman's rank correlation test. The associations between mRNA or protein expression \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003elevels\u003c/span\u003e of ATP7B and clinicopathological factors were analyzed using \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ethe\u003c/span\u003e χ2 test. Disease-free survival (DFS) and OS were calculated using the Kaplan-Meier method, and survival curves were compared using the log-rank test. Multivariate analysis was performed using the Cox hazard model. All statistical analyses were performed using JMP 16 software (SAS Institute Inc., Cary, NC, USA), and statistical significance was defined as \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eATP7B\u003c/b\u003e \u003cb\u003emRNA expression and its association with other cancer-related genes in BC cell lines\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe mRNA expression levels of \u003cem\u003eATP7B\u003c/em\u003e in 13 BC cell lines and two non-cancerous cell lines are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea. The ER, PgR, and HER2 statuses of cell lines have been evaluated in previous studies [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. \u003cem\u003eATP7B\u003c/em\u003e mRNA levels in ER-positive and HER2-positive cell lines were significantly higher than those in ER-negative and HER2-negative BC cells (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.015 and \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.028, respectively). To compensate for the small number of cell lines, \u003cem\u003eATP7B\u003c/em\u003e mRNA expression levels in \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eadditional\u003c/span\u003e BC cell lines were obtained from the CCLE database for verification. The ER, PgR, and HER2 statuses of each cell line were obtained from previous studies [\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Among the 60 BC cells with available data regarding \u003cem\u003eATP7B\u003c/em\u003e expression levels, ER-positive, PgR-positive, and HER2-positive cells had significantly higher \u003cem\u003eATP7B\u003c/em\u003e mRNA levels than their negative counterparts (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.002, and \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.040, respectively; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePCR array analysis revealed that \u003cem\u003eATP7B\u003c/em\u003e mRNA expression levels were positively correlated with those of several well-known oncogenes, such as \u003cem\u003ecadherin 1\u003c/em\u003e (\u003cem\u003eCDH1\u003c/em\u003e) and \u003cem\u003eestrogen receptor 1\u003c/em\u003e (\u003cem\u003eESR1\u003c/em\u003e), and negatively correlated with \u003cem\u003eMET proto-oncogene\u003c/em\u003e (\u003cem\u003eMET\u003c/em\u003e) (Tables\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and S1).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCorrelations between mRNA expression levels of \u003cem\u003eATP7B\u003c/em\u003e and cancer-related genes.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGene\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOfficial Full Name\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCorrelation\u003c/p\u003e \u003cp\u003eCoefficient\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003ep\u003c/em\u003e-value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCDH1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCadherin 1, type 1, E-cadherin (epithelial)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.863\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eESR1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEstrogen receptor 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.709\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.007\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eRET\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRet proto-oncogene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.681\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.010\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eZHX2\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eZinc fingers and homeoboxes 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.632\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.021\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eMYB\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eV-myb myeloblastosis viral oncogene homolog (avian)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.615\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.025\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eMYCN\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eV-myc myelocytomatosis viral related oncogene, neuroblastoma derived\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.615\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.025\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eMET\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMet proto-oncogene (hepatocyte growth factor receptor)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u0026minus;0.681\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.010\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eJUN\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eJun\u0026nbsp;proto-oncogene, AP-1 transcription factor subunit\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u0026minus;0.615\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.025\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eETS1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eV-ets erythroblastosis virus E26 oncogene homolog 1 (avian)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u0026minus;0.610\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.027\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ePML\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePromyelocytic leukemia\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u0026minus;0.593\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.033\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eTGFB1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTransforming growth factor, beta 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u0026minus;0.566\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.044\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eEffects of ATP7B knockdown in BC cell lines\u003c/h2\u003e \u003cp\u003eWestern blotting was performed using representative BC cell lines with high or low \u003cem\u003eATP7B\u003c/em\u003e mRNA expression to confirm the protein expression of ATP7B. Among these cell lines, MDA-MB-361 represents the ER-positive/HER2-positive subtype, and MDA-MB-415 represents the ER-positive/HER2-negative subtype. MDA-MB-231, one of the cell lines with the lowest \u003cspan type=\"ItalicUnderline\" class=\"ItalicUnderline\" name=\"Emphasis\"\u003eATP7B\u003c/span\u003e mRNA expression, was used as a negative control (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). Cells transfected with siRNA expressed lower \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003elevels of ATP7B\u003c/span\u003e mRNA and protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed and e).\u003c/p\u003e \u003cp\u003eTo determine the oncological role of ATP7B in BC cells, cell proliferation, invasiveness, and migration were evaluated using knockdown cells. During the entire study period, proliferation was significantly enhanced in si\u003cem\u003eATP7B\u003c/em\u003e-transfected MDA-MB-361 and MDA-MB-415 cells compared to that in untransfected and siControl-transfected cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). In the invasiveness assay, more si\u003cem\u003eATP7B\u003c/em\u003e-transfected than siControl-transfected or untransfected MDA-MB-361 and MDA-MB-415 cells passed through the Matrigel (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). Moreover, the migratory abilities of MDA-MB-361 and MDA-MB-415 cells were enhanced following si\u003cem\u003eATP7B\u003c/em\u003e transfection (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eAssociation between\u003c/b\u003e \u003cb\u003eATP7B\u003c/b\u003e \u003cb\u003emRNA expression levels and clinicopathological factors\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003eATP7B\u003c/em\u003e mRNA expression levels were evaluated in both BC and non-cancerous specimens. The ratio of \u003cem\u003eATP7B\u003c/em\u003e mRNA expression levels between cancerous and non‑cancerous specimens was defined as the \u0026lsquo;C/N ratio.\u0026rsquo; The mean C/N ratio (\u0026plusmn;\u0026thinsp;SD) was 1.28\u0026thinsp;\u0026plusmn;\u0026thinsp;1.64, with 69 (44.2%) patients having a C/N ratio greater than one. The \u003cem\u003eATP7B\u003c/em\u003e C/N ratios were not predominant in the T category, lymph node metastasis, or UICC stage (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Regarding conventional biomarkers, ER-positive specimens (n\u0026thinsp;=\u0026thinsp;119) had a higher \u003cem\u003eATP7B\u003c/em\u003e C/N ratio than ER-negative specimens (n\u0026thinsp;=\u0026thinsp;37; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and PgR-positive specimens (n\u0026thinsp;=\u0026thinsp;108) had significantly higher \u003cem\u003eATP7B\u003c/em\u003e C/N ratios than PgR-negative specimens (n\u0026thinsp;=\u0026thinsp;48; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.003; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). The \u003cem\u003eATP7B\u003c/em\u003e C/N ratio \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003edid not differ significantly\u003c/span\u003e between HER2-positive (n\u0026thinsp;=\u0026thinsp;37) and HER2-negative specimens (n\u0026thinsp;=\u0026thinsp;111; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.279; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePatients with a C/N ratio greater than one were assigned to the \u0026ldquo;high \u003cem\u003eATP7B\u003c/em\u003e group\u0026rdquo; (n\u0026thinsp;=\u0026thinsp;69), while those with a C/N ratio less than one were assigned to the \u0026ldquo;low \u003cem\u003eATP7B\u003c/em\u003e group\u0026rdquo; (n\u0026thinsp;=\u0026thinsp;87). The associations between clinicopathological factors and \u003cem\u003eATP7B\u003c/em\u003e expression are shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Tumor size, lymph node metastasis, or UICC pathological stage did not \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ediffer significantly\u003c/span\u003e between the two groups. The high \u003cem\u003eATP7B\u003c/em\u003e group had more ER-positive and PgR-positive patients than the low \u003cem\u003eATP7B\u003c/em\u003e group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.002 and \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.030, respectively).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAssociations between \u003cem\u003eATP7B\u003c/em\u003e mRNA expression and the clinicopathological characteristics of 156 patients with breast cancer\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eCharacteristics\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eExpression of \u003cem\u003eATP7B\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003ep\u003c/em\u003e-value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHigh \u003cem\u003eATP7B\u003c/em\u003e group (n\u0026thinsp;=\u0026thinsp;69)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLow \u003cem\u003eATP7B\u003c/em\u003e group (n\u0026thinsp;=\u0026thinsp;87)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge (range)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e50 (26\u0026ndash;78)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e54 (30\u0026ndash;77)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.024\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHistology\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.138\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDCIS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5(7.3%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1(1.1%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIDC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e57(82.6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e81(93.1%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eILC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4(5.8%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2(2.3%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOther\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3(4.3%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3(3.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUICC T category\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.050\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTis/T1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e37(53.6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e33(37.9%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT2/T3/T4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e32(46.4%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e54(62.1%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLymph node status\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.147\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePositive\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e30(43.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e48(55.2%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e39(56.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e39(44.8%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUICC pathological stage\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.133\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0/I\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e26(37.7%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e23(26.4%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eII/III/IV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e43(62.3%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e64(73.6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eER status\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.002\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePositive\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e61(88.4%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e58(66.7%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8(11.6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e29(33.3%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePgR status\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.030\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePositive\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e54(78.3%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e54(62.1%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e15(21.7%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e33(37.9%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHER2 status\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.176\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePositive\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e12(17.4%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e25(28.7%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e52(75.4%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e59(67.8%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUnknown\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5(2.2%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3(3.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAdjuvant therapy\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.101\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEndocrine therapy alone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e28(40.6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e22(25.3%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eChemotherapy alone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8(11.6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e21(24.1%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEndocrine and chemotherapy\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e26(37.7%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e36(41.4%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7(10.1%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8(9.2%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e \u003cp\u003eData are expressed as the median (range) or number (%). \u003csup\u003ea\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05. ATP7B, ATPase Copper Transporting Beta; DCIS, ductal carcinoma \u003cem\u003ein situ\u003c/em\u003e; ER, estrogen receptor; HER2, human epidermal growth factor 2; IDC, invasive ductal carcinoma; ILC, invasive lobular carcinoma; PgR, progesterone receptor; Tis, tumor \u003cem\u003ein situ\u003c/em\u003e; UICC, Union for International Cancer control\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe high \u003cem\u003eATP7B\u003c/em\u003e group had a significantly longer DFS than the low \u003cem\u003eATP7B\u003c/em\u003e group (5-year DFS, high \u003cem\u003eATP7B\u003c/em\u003e group: 92.7%, low \u003cem\u003eATP7B\u003c/em\u003e group: 76.6%; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.004; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). The OS rates in the high \u003cem\u003eATP7B\u003c/em\u003e group were also longer than that in the low \u003cem\u003eATP7B\u003c/em\u003e group (5-year OS: high \u003cem\u003eATP7B\u003c/em\u003e group, 92.8%; low \u003cem\u003eATP7B\u003c/em\u003e group, 88.4%; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.019; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). To compensate for the small number of patients in our cohort, the prognostic value of \u003cem\u003eATP7B\u003c/em\u003e expression was validated using the Kaplan-Meier plotter website. Similarly, when patients were \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eseparated based on the\u003c/span\u003e median \u003cem\u003eATP7B\u003c/em\u003e expression, the high \u003cem\u003eATP7B\u003c/em\u003e expression group exhibited significantly longer RFS (n\u0026thinsp;=\u0026thinsp;4929; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and OS (n\u0026thinsp;=\u0026thinsp;1879; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.013) (Fig.\u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e a \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eand\u003c/span\u003e b). Multivariate analysis of DFS revealed \u0026lsquo;lymph node metastasis\u0026rsquo; (HR, 3.56; 95% CI, 1.42\u0026ndash;8.92; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.007) and \u0026lsquo;low \u003cem\u003eATP7B\u003c/em\u003e expression\u0026rsquo; (HR, 2.82; 95% CI, 1.15\u0026ndash;6.92; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.024) as independent prognostic factors (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePrognostic factors for disease-free survival in 156 breast cancer patients.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eVariable\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003en\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e \u003cp\u003eUnivariate\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c9\" namest=\"c7\"\u003e \u003cp\u003eMultivariate\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHazard ratio\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e95% CI\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003ep\u003c/em\u003e-value\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eHazard ratio\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003e95% CI\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cem\u003ep\u003c/em\u003e-value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge, \u0026gt;\u0026thinsp;60 years\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.48\u0026ndash;2.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.948\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTumor size, \u0026gt;2cm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.51\u0026ndash;8.99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.004\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.91\u0026ndash;5.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.081\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNode status, positive\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.05\u0026ndash;12.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e3.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.42\u0026ndash;8.92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.007\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eER status, negative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.86\u0026ndash;3.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.120\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePgR status, negative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.77\u0026ndash;3.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.210\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHER2 status, positive\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.92\u0026ndash;4.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.082\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLow \u003cem\u003eATP7B\u003c/em\u003e expression\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.50\u0026ndash;8.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.004\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.15\u0026ndash;6.92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.024\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"9\" nameend=\"c9\" namest=\"c1\"\u003e \u003cp\u003e\u003csup\u003ea\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05. Univariate analysis: Cox proportional hazards model. Multivariate analysis: Cox proportional hazards model. CI, confidence interval; ER, estrogen receptor; HER2, human epidermal growth factor 2; PgR, progesterone receptor\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eAssessment of ATP7B protein expression status\u003c/b\u003e \u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eby\u003c/span\u003e \u003cb\u003eimmunohistochemistry\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAt the protein level, cytoplasmic ATP7B staining was evaluated \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eby\u003c/span\u003e immunohistochemistry in 152 BC specimens. Representative staining of the IS and PS of ATP7B is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea. Patients with an IP score (IS x PS score) of 150 or higher were assigned to the \u0026lsquo;high ATP7B group\u0026rsquo; (n\u0026thinsp;=\u0026thinsp;73) while the remaining patients were assigned to the \u0026lsquo;low ATP7B group\u0026rsquo; (n\u0026thinsp;=\u0026thinsp;79). The \u003cem\u003eATP7B\u003c/em\u003e C/N ratio was higher in the high ATP7B group than in the low ATP7B group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.026; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb), which validated the consistency between the mRNA and protein levels. Although DFS \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003edid not differ\u003c/span\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec), the OS rates were significantly higher in the high ATP7B group than in the low ATP7B group (5-year OS: high ATP7B group, 94.5%; low ATP7B group, 86.1%; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.041; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). Multivariate analysis of OS revealed \u0026lsquo;lymph node metastasis\u0026rsquo; (HR, 4.71; 95% CI, 1.75\u0026ndash;12.7; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.002), \u0026lsquo;ER negative\u0026rsquo; (HR, 7.92; 95% CI, 1.01\u0026ndash;61.8; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.048), and \u0026lsquo;low ATP7B expression\u0026rsquo; (HR, 2.38; 95% CI, 1.02\u0026ndash;5.57; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.046) as independent prognostic factors (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, ATP7B interfered with tumor progression by suppressing the proliferation, invasiveness, and migration of BC cells. Furthermore, the analysis of clinical specimens revealed that ATP7B mRNA and protein expression were independent prognostic factors, \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ethus\u003c/span\u003e supporting the \u003cem\u003ein vitro\u003c/em\u003e results.\u003c/p\u003e \u003cp\u003eATP7B is a member of the \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eP\u003c/span\u003e-type ATPase family and is involved in intracellular copper transport and homeostasis [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Notably, \u003cem\u003eATP7B\u003c/em\u003e is one of the genes that has attracted \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003econsiderable\u003c/span\u003e attention for its potential involvement in cuproptosis [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. According to a recent study, \u003cem\u003eATP7B\u003c/em\u003e is differentially expressed in various carcinomas, suggesting a prognostic implication for patients with low-grade glioma and renal clear cell carcinoma [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. \u003cem\u003eATP7B\u003c/em\u003e may also serve as a therapeutic target to improve the efficacy of docetaxel in prostate cancer [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. In BC, \u003cem\u003eATP7B\u003c/em\u003e induces resistance to cisplatin [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. High \u003cem\u003eATP7B\u003c/em\u003e expression in patients with ER-positive BC \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ehas also been\u003c/span\u003e reported to be associated with a lower risk of relapse [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. However, the oncological role and significance of ATP7B in patients have not been elucidated \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eto date\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eIn this study, \u003cem\u003eATP7B\u003c/em\u003e was highly expressed in ER-positive cell lines, consistent with the results obtained from the CCLE database. Furthermore, our clinical specimens from ER-positive patients with BC were found to have high expression of \u003cem\u003eATP7B\u003c/em\u003e compared to those from ER-negative patients. Based on PCR array analysis, the expression level of \u003cem\u003eESR1\u003c/em\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ecorrelated positively\u003c/span\u003e with that of \u003cem\u003eATP7B\u003c/em\u003e, \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ethus\u003c/span\u003e supporting these observational results. A previous study did not \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003efind\u003c/span\u003e a significant relationship between \u003cem\u003eATP7B\u003c/em\u003e and ER-positivity in patients with BC [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]; \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ethis\u003c/span\u003e finding may be due to the small number of samples (41 patients), which resulted in insufficient statistical power. \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eThe consistency of our results with cell lines and clinical samples implies\u003c/span\u003e that ATP7B is involved in the ER signaling pathway in BC. However, further pathway analyses are required to clarify the role of ATP7B \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ein this regard\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eOverall, knockdown of ATP7B was found to promote malignant phenotypes in BC cells and patients with high ATP7B expression had a favorable prognosis in this study. The expression status of ATP7B was also identified as an independent prognostic factor at both the mRNA and protein levels, leading to more robust results. Although functional analysis of ATP7B in BC cells and the prognostic impact of its expression in patients with BC have not been previously assessed, a recent study revealed the association between cuproptosis-related gene expression and prognosis in ER-positive patients with BC [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Accordingly, patients with high expression of some cuproptosis-related genes, including \u003cem\u003eATP7B\u003c/em\u003e, had favorable relapse-free survival \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003erates\u003c/span\u003e. Although these results are similar to those of our study, we have demonstrated that ATP7B alone can be used as a prognostic marker for BC, regardless of subtypes.\u003c/p\u003e \u003cp\u003eAlthough functional analyses revealed that ATP7B plays tumor-suppressive roles in BC cells, elucidating the underlying mechanism remains a difficult task. Nevertheless, based on the results of our PCR array analysis of BC cells and those of previous studies, several explanations can be proposed. The highest correlation coefficient was observed between the expression levels of \u003cem\u003eATP7B\u003c/em\u003e and \u003cem\u003eCDH1\u003c/em\u003e, which encodes E-cadherin. E-cadherins contribute to the establishment and maintenance of polarized and differentiated epithelia through intercellular adhesion complexes, and their inactivation increases \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ethe metastatic capacity, which leads to poor prognoses\u003c/span\u003e in BC [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. In contrast, \u003cem\u003eATP7B\u003c/em\u003e expression negatively correlated with \u003cem\u003eMET\u003c/em\u003e expression. \u003cem\u003eMET\u003c/em\u003e encodes the tyrosine kinase receptor for hepatocyte growth factor, and its activation promotes cellular invasiveness, angiogenesis, and metastasis [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Although no studies \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eto date\u003c/span\u003e have suggested a direct relationship between ATP7B and these molecules, ATP7B may affect the expression of these genes to suppress tumor progression in BC.\u003c/p\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eNotably, analysis of the clinical samples identified both the mRNA and protein levels of ATP7B as prognostic markers. The cut-off values of ATP7B expression levels have not been established to date. In this study, in terms of the mRNA expression levels, patients with a C/N ratio greater than one were assigned to the \u0026ldquo;high\u003c/span\u003e \u003cspan type=\"ItalicUnderline\" class=\"ItalicUnderline\" name=\"Emphasis\"\u003eATP7B\u003c/span\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003egroup\u0026rdquo; (n\u0026thinsp;=\u0026thinsp;69) while those with a C/N ratio less than one were assigned to the \u0026ldquo;low\u003c/span\u003e \u003cspan type=\"ItalicUnderline\" class=\"ItalicUnderline\" name=\"Emphasis\"\u003eATP7B\u003c/span\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003egroup\u0026rdquo; (n\u0026thinsp;=\u0026thinsp;87). This grouping method is considered relatively reasonable. On the other hand, for the immunohistochemistry, we grouped the patients according to the IP score. Patients with an IP score of 150 or higher were then assigned to the \u0026lsquo;high ATP7B group\u0026rsquo; (n\u0026thinsp;=\u0026thinsp;73) while the remaining patients were assigned to the \u0026lsquo;low ATP7B group\u0026rsquo; (n\u0026thinsp;=\u0026thinsp;79). This cut-off value allocated almost the same number of cases as the grouping at the mRNA expression levels. Moreover, because\u003c/span\u003e \u003cspan type=\"ItalicUnderline\" class=\"ItalicUnderline\" name=\"Emphasis\"\u003eATP7B\u003c/span\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003emRNA expression was shown to be higher in \u0026lsquo;high ATP7B group\u0026rsquo;, this criterion closely reflected the grouping at the mRNA level. Although our results are supported by the public database, further validation studies will be required to determine appropriate cut-off values.\u003c/span\u003e\u003c/p\u003e \u003cp\u003eThis study had several limitations. As described above, the mechanism by which ATP7B is involved in tumor suppression has not been fully investigated, despite the proposal of several theories. In particular, the mechanism by which ATP7B affects copper transport in BC cells to attenuate their malignant features remains to be elucidated. Second, adjuvant medication therapy \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ecould\u003c/span\u003e have affected \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ethe\u003c/span\u003e patient prognoses in our cohort data. To compensate for this effect, we used a public database to validate our results. \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eIn addition, further studies, such as\u003c/span\u003e \u003cspan type=\"ItalicUnderline\" class=\"ItalicUnderline\" name=\"Emphasis\"\u003ein vivo\u003c/span\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eexperiments, are warranted to elucidate the potential therapeutic targets of ATP7B.\u003c/span\u003e\u003c/p\u003e \u003cp\u003eIn conclusion, this study revealed the tumor-suppressive roles of ATP7B in BC cells. Moreover, this study highlighted that high ATP7B expression in cancerous tissues could be a favorable prognostic biomarker in patients with BC.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo funding was received.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of Data and Materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed in the current study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors and Affiliations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDepartment of Breast and Endocrine Surgery, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 4668560, Japan\u003c/p\u003e\n\u003cp\u003eIkumi Soeda, Masahiro Shibata, Takahiro Ichikawa, Kayoko Sugino, \u0026amp; Norikazu Masuda\u003c/p\u003e\n\u003cp\u003eDepartment of Surgery, Nagoya Ekisaikai Hospital, 4-66, Shonen-cho, Nakagawa-ku, Nagoya 4548502, Japan\u003c/p\u003e\n\u003cp\u003eMasahiro Shibata\u003c/p\u003e\n\u003cp\u003eDepartment of Surgery, Komaki City Hospital, 1-20, Joubushi, Komaki Aichi 4858520, Japan\u003c/p\u003e\n\u003cp\u003eTakahiro Inaishi\u003c/p\u003e\n\u003cp\u003eDepartment of Surgery and Science, Faculty of Medicine, Academic Assembly, University of Toyama 2630 Sugitani, Toyama-shi, Toyama, 930-0194, Japan\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eEmi Kanaya\u003c/p\u003e\n\u003cp\u003eDepartment of Gastroenterological Surgery, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 4668560, Japan\u003c/p\u003e\n\u003cp\u003eMitsuro Kanda \u0026amp; Masamichi Hayashi\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIS, MS, and MK conceived and designed the study. IS, MS, TIn, and TIc conducted the experiments. IS and MS analyzed the data and wrote the manuscript. MS, TIn, and TIc contributed to \u003cu\u003ethe\u003c/u\u003e acquisition of patient data. TIn, TIc, KS, EK, MK, MH, and NM contributed to the interpretation of comprehensive data and revised the manuscript for important intellectual content. All authors have read and approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and Consent to Participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe present study was approved by the Institutional Review Board and Ethics Committee of Nagoya University Hospital (approval no. 2019‑0028). Informed consent was obtained from all patients included in the study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePatient consent for Publication\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe participants provided written informed consent for publication as required by the Institutional Review Board and Ethics Committee of Nagoya University Hospital.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNorikazu Masuda received a research grant from Chugai Pharma, Eli Lilly Japan, AstraZeneca, Pfizer, Daiichi Sankyo, MSD, Eisai, Novartis, Gilead Sciences, and Ono Pharma; received lecture fees from Chugai Pharma, Pfizer, AstraZeneca, Eli Lilly Japan, Daiichi Sankyo, and Eisai; and is a board representative of the Japan Breast Cancer Research Group (JBCRG) and a board member of the Japanese Breast Cancer Society (JBCS),\u003cu\u003e\u0026nbsp;the\u003c/u\u003e Japan Society of Clinical Oncology (JSCO), and \u003cu\u003ethe\u003c/u\u003e Japan Association of Breast Cancer Screening (JABCS), all without remuneration. \u003cu\u003eThe\u003c/u\u003e other authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSiegel RL, Miller KD, Wagle NS, Jemal A. Cancer statistics, 2023. CA Cancer J Clin. 2023;73:17\u0026ndash;48.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePalmgren MG, Nissen P. P-type ATPases. Annu Rev Biophys. 2011;40:243\u0026ndash;66.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePolishchuk EV, Merolla A, Lichtmannegger J, Romano A, Indrieri A, Ilyechova EY et al. Activation of Autophagy, Observed in Liver Tissues From Patients With Wilson Disease and From ATP7B-Deficient Animals, Protects Hepatocytes From Copper-Induced Apoptosis. Gastroenterology. 2019; 156: 1173-89 e5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi YQ, Yin JY, Liu ZQ, Li XP. Copper efflux transporters ATP7A and ATP7B: Novel biomarkers for platinum drug resistance and targets for therapy. IUBMB Life. 2018;70:183\u0026ndash;91.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eInesi G, Pilankatta R, Tadini-Buoninsegni F. Biochemical characterization of P-type copper ATPases. 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PLoS ONE. 2013;8:e77232.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNeve RM, Chin K, Fridlyand J, Yeh J, Baehner FL, Fevr T, et al. A collection of breast cancer cell lines for the study of functionally distinct cancer subtypes. Cancer Cell. 2006;10:515\u0026ndash;27.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang Z, Zhang A, Shi Y, Zhao Z, Zhao Z. An association between ATP7B expression and human cancer prognosis and immunotherapy: a pan-cancer perspective. BMC Med Genomics. 2023;16:307.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSong L, Nguyen V, Xie J, Jia S, Chang CJ, Uchio E, et al. ATPase Copper Transporting Beta (ATP7B) Is a Novel Target for Improving the Therapeutic Efficacy of Docetaxel by Disulfiram/Copper in Human Prostate Cancer. Mol Cancer Ther. 2024;23:854\u0026ndash;63.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFan Y, Luo C, Wang Y, Wang Z, Wang C, Zhong X, et al. A nomogram based on cuproptosis-related genes predicts 7-year relapse-free survival in patients with estrogen receptor-positive early breast cancer. Front Oncol. 2023;13:1111480.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCorso G, Figueiredo J, De Angelis SP, Corso F, Girardi A, Pereira J, et al. E-cadherin deregulation in breast cancer. J Cell Mol Med. 2020;24:5930\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGastaldi S, Comoglio PM, Trusolino L. The Met oncogene and basal-like breast cancer: another culprit to watch out for? Breast Cancer Res. 2010;12:208.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"breast-cancer","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"brca","sideBox":"Learn more about [Breast Cancer](http://link.springer.com/journal/12282)","snPcode":"12282","submissionUrl":"https://www.editorialmanager.com/brca/default2.aspx","title":"Breast Cancer","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"ATPase copper transporting beta, breast cancer, estrogen receptor, prognostic marker","lastPublishedDoi":"10.21203/rs.3.rs-6101234/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6101234/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground.\u003c/strong\u003e ATPase copper transporting beta (ATP7B) functions as a copper-transporting ATPase that ejects copper from cells. Although high expression of ATP7B has been reported to increase cisplatin resistance, its role in breast cancer (BC) remains unclear. This study aimed to elucidate the function of ATP7B in BC cells and its significance in patients with BC.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods.\u003c/strong\u003e The mRNA and protein expression levels of ATP7B were evaluated in BC and non-cancerous mammary cell lines. Polymerase chain reaction (PCR) array analysis was conducted to determine the correlation between \u003cem\u003eATP7B\u003c/em\u003e and 84 cancer-related genes. \u003cem\u003eATP7B\u003c/em\u003e knockdown was performed using small interfering RNA, and cell proliferation, invasiveness, and migration were analyzed. The associations between the mRNA and protein expression of ATP7B and clinicopathological factors were also investigated in 156 patients with BC.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults.\u003c/strong\u003e \u003cem\u003eATP7B\u003c/em\u003ewas found to be highly expressed in estrogen receptor-positive and human epidermal growth factor receptor 2-positive BC cell lines. PCR array analysis revealed a significant correlation between the expression level of \u003cem\u003eATP7B\u003c/em\u003e and those of cadherin 1, estrogen receptor 1, and MET proto-oncogene. \u003cem\u003eATP7B\u003c/em\u003e knockdown significantly increased the proliferation, invasiveness, and migration of MDA-MB-361 and MDA-MB-415 cells. Patients with high ATP7B expression at the mRNA and protein levels experienced favorable prognoses. In addition, ATP7B expression level was identified as an independent prognostic factor in multivariate analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions.\u003c/strong\u003e ATP7B is involved in promoting anti-cancer activities of tumor suppressors in BC cells across different subtypes and is considered a prognostic marker for BC.\u003c/p\u003e","manuscriptTitle":"ATPase copper transporting beta attenuates malignant features with high expression as an indicator of favorable prognosis in breast cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-17 08:39:50","doi":"10.21203/rs.3.rs-6101234/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Accept","date":"2025-04-14T21:15:22+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2025-04-10T04:00:14+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-10T03:58:16+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-04-10T03:51:06+00:00","index":"","fulltext":""},{"type":"submitted","content":"Breast Cancer","date":"2025-04-09T08:42:40+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"breast-cancer","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"brca","sideBox":"Learn more about [Breast Cancer](http://link.springer.com/journal/12282)","snPcode":"12282","submissionUrl":"https://www.editorialmanager.com/brca/default2.aspx","title":"Breast Cancer","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"5d33e80c-a522-41e2-a116-0b798e7eb4c0","owner":[],"postedDate":"April 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-05-05T16:01:24+00:00","versionOfRecord":{"articleIdentity":"rs-6101234","link":"https://doi.org/10.1007/s12282-025-01705-7","journal":{"identity":"breast-cancer","isVorOnly":false,"title":"Breast Cancer"},"publishedOn":"2025-05-02 15:57:37","publishedOnDateReadable":"May 2nd, 2025"},"versionCreatedAt":"2025-04-17 08:39:50","video":"","vorDoi":"10.1007/s12282-025-01705-7","vorDoiUrl":"https://doi.org/10.1007/s12282-025-01705-7","workflowStages":[]},"version":"v1","identity":"rs-6101234","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6101234","identity":"rs-6101234","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}